Interferon (IFN) plays a central role in host intrinsic immunity to viral infection, the underlying mechanism of which remains poorly defined. Towards this goal, we have recently shown that interferon-induced transmembrane (IFITM) proteins profoundly inhibit the membrane fusion and infection of a number of enveloped viruses, including HIV-1. Interestingly, we found that, while human IFITM2 and IFITM3 impede HIV- 1 (BH10) entry, human IFITM1 impairs viral infectivity. Notably, the prolonged culture of BH10 HIV-1 led to the emergence of mutations in HIV-1 Env that render the virus resistant to IFITM1 inhibition, suggesting that IFITMs may functionally act on HIV-1 Env and diminish viral infectivity. The goal of this R01 project is to determine the mechanisms by which IFITM proteins inhibit distinct steps of HIV replication, as well as viral antagonisms. Aim 1 will address how IFITM proteins inhibit HIV-1 entry. We will use novel cell-cell fusion and single virus fusion techniques to test the hypothesis that both hemifusion and pore expansion are inhibited by IFITM2 and IFITM3. Aim 2 will focus on how IFITM proteins, especially IFITM1, diminish HIV-1 infectivity. We will test the central hypothesis that IFITM proteins are incorporated into HIV-1 particles and functionally inactivate HIV-1 Env activity. Aim 3 will characterize HIV-1 antagonisms against IFITMs, particularly the possible role of HIV-1 Env in this process. Collectively, results from this project will provide critical insights into the mechanisms of actio of IFITMs, and will aid in the development of novel antiviral agents against HIV-1 infection.